Contaminant reducing amide functionalized ordered mesoporous carbon composition

ABSTRACT

The present invention is directed to compositions and methods related to delivering degradative enzymes to remove/remedy environmental pollutants. The inventive material comprises a series of amide-functionalized ordered mesoporous carbon (AFOMC), which utilizes chemical conjugation techniques for the tethering of enzymes to the surface of the synthesized AFOMC. The delivery mechanism may be utilized to express a wide variety of toxin-degrading enzymes for removal/remediation of organic pollutants.

RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/517,189, filed on Apr. 15, 2011 the teachings and contents ofwhich are incorporated herein by reference.

SEQUENCE LISTING

This application contains a sequence listing in computer readableformat, the teachings and content of which are hereby incorporated byreference.

FIELD OF INVENTION

The present invention relates to compounds and methods for remediationof environmental pollutants, more specifically, to a series ofamide-functionalized ordered mesoporous carbon for delivery oftoxin-degrading enzymes for removal/remediation of organic pollutants.

BACKGROUND OF INVENTION

Atrazine (ATR,2-chloro-4-(ethylamino)-6-(isopropyl-amino)-1,3,5-triazine) has been oneof the most widely applied herbicides in the US and Midwestern states.An estimated 36.3 million kg of ATR is applied annually to more than 69%of all U.S. corn acreage (United States Department of Agriculture,2004). The contamination of surface and ground water by ATR and itschlorinated metabolites has raised public health and ecological concernsrecently due to their endocrine disruption activities and potential riskof gastroschisis (Hayes et al., 2006; Waller et al., 2010). The ATR andits metabolites are persistent in the environmental and themineralization of ATR and its chlorinated metabolites or completecleavage of the triazine ring in the environment is limited to less than2-10%. A 2006 study by the U.S. Geological Survey found ATR and itsmetabolites were detected in approximately 75 percent of stream waterand about 40 percent of all groundwater samples from agricultural areastested between 1992 and 2001 (Gilliom et al., 2006).

Despite the persistence of ATR and its metabolites in the VBSenvironment (Lin et al., 2005), the enzymes including chlorohydrolaseAtzA produced by naturally occurring degradative bacteria Pseudomonassp. strain ADP rapidly hydrolyzes the atrazine to the much less toxicand less mobile metabolite hydroxyatrazine. The catabolic genes thatencode for the enzymes responsible for each step in the ATR degradationprocess have been well characterized (FIG. 1). Pseudomonas sp. strainADP can utilize ATR and its metabolites as a carbon source and solenitrogen source (Mandelbaum et al., 1995; Martinez et al., 2001). Thisproperty is due to the presence of the pADP-1 plasmid, a 108-kDAcatabolic plasmid which encodes for all the metabolic enzymes necessaryto completely degrade ATR to CO₂ and NH₃ (De Souza et al., 1998a;Martinez et al., 2001). The AtzA-C enzymes are not unique to Pseudomonassp. strain ADP, but are found among soil bacteria isolates across theU.S. and Europe (De Souza et al., 1998b). The atzA chlorohydrolasemetalloenzyme not only has the ability to dechlorinate atrazine into thesignificantly less toxic hydroxyatrazine, but also shows degradativeactivity to other s-triazines, such as simazine and desethylatrazine(Boundy-Mills et al., 1997; De Souza et al., 1996). Catabolic gene atzBmetabolizes hydroxyatrazine to N-isopropylammelide, whose hydrolyticdeamidation to cyanuric acid and isopropylamine is catalyzed by atzC(Boundy-Mills et al., 1997; Sadowsky et al., 1998). Gene atzD encodes acyanuric acid amidohydrolase, which converts cyanuric acid to biuret.The presence of the atzDEF operon is unique to Pseudomonas sp. strainADP and allows this bacterium to further catabolize the cyanuric acid(produced by the activities of AtzA-C) into CO₂ and NH₃ (Martinez etal., 2001). Biuret hydrolase is encoded by atzE and allophanatehydrolase is encoded by atzF, resulting in the conversion of biuret toallophanate and allophanate to CO₂ and NH₃ (Martinez et al., 2001). Thisplasmid is highly transmittable between microorganisms, and theexpression of these genes, particularly atzD and F, are sensitive toalternative N sources in the environment (atz+ to atz−) (Garcia-Gonzalezet al., 2003). The purification and optimization of ATR enzymes forlarge scale remediation of organic pesticides including atrazine werefirst commercialized by CSIRO Enzyme Based Bioremediation Technology.However, the persistence of the enzymes such as AtzN has short lifeunder the field conditions. The enzymatic activities cannot be sustainedmore than 8-10 days.

The recent success of enzyme conjugation technology and orderedmesoporous material synthesis (Hartmann, 2005) has allowed theencapsulation of enzymes and other biomolecules into ordered mesoporousmaterial. According to IUPAC definition, material containing pores withdiameters ranging from 2 to 50 nm are classified as ordered mesoporousmaterial. The modified ordered mesoporous carbon material have highspecific surface area, large specific pore volume, regular arrays ofuniform nanopores, and narrow pore size of distribution which provide alarge adsorption capacities and unique temples for functionalmodification. To immobilize the bioactive biomolecules or enzymes on thesurface of the ordered mesoporous material, the surface of porousmaterials usually needs to be modified and functionalized. Thefunctionalization of the materials usually carries out by reacting withorganic or inorganic oxidative agents, such as nitric acid, ozone, orammonium persulfate, followed by the substitution of oxidative groups byfunctionalities containing amine (—NH₂), thiol (R—SH) or free sulfhydryl(—SH) (Contescu, 1998; Hartmann, 2005; Jarrais, 2005; Puri, 1971; Tamai,2006a). The functionalization processes provide the functional groupsrequired for conjugation of biomolecules or enzymes. Hypothetically, theordered mesoporous materials immobilized with bioactive enzymes orbiomolecules should not only retain their functional enzymatic orbiological characteristics by the enzymes/biomolecules immobilized onthe surface, but also exhibit high specific surface areas and largeadsorption capacities for the organic pollutants, like atrazine, throughelectrostatic interactions and/or covalent bindings.

Therefore, there is a need to provide a new and improved series ofenzymatic delivery compounds with improved surface specificity andadsorption capacity and the ability to retain the enzymes' biologicalcharacteristics during delivery.

SUMMARY OF INVENTION

The present invention provides a new and improved series ofamide-functionalized ordered mesoporous carbon (AFOMC) as a vehicle andsystem to deliver enzymes that degrade pollutants or toxins. The systemis suitable for the production and delivery of materials such asbioparticles, proteins and small molecules. Preferably, the AFOMC of thepresent invention utilize atrazine-degrading enzymes, including but notlimited to AtzA, AtzB, AtzC, AtzD, AtzE, AtzF, and combinations of theseenzymes. The AFOMC of the present invention have a variety ofapplications, including, but not limited to, a biofilter, a kit forremoving pollutants or toxins, biocatalysts, fuel cells, imaging,biofuel production, and biosensors. A method for removing pollutants ortoxins from water and soil sources is also disclosed. The methodincludes the steps of introducing the AFOMC into a water or soil sourceand allowing the AFOMC to enhance both adoption and degradation ofpollutants and, optionally, to allow the tethered enzymes to persistover time. The present invention utilizes chemical conjugationtechniques for the tethering of enzymes to the surface of thesynthesized AFOMC. The delivery mechanism may be utilized to express awide variety of toxin-degrading enzymes for removal/remediation of theorganic pollutants. In addition, this conjugation of bioactive enzymesonto the amide-functionalized ordered mesoporous carbon have a widerange of other commercial applications ranging from development ofbiocatalysts, biofilters, fuel cells, drug delivery systems, othermedical therapeutics and bio sensors.

DEFINITIONS

“Pollutants” and “Toxins”, for purposes of the present invention,include any undesirable substance found within a material or liquid. Apollutant or toxin may be found in air, gas, liquid, gel, soil, plants,food materials, water sources, and combinations thereof. This list isnot meant to be limiting, as any undesirable element found in a sourcecould be used for purposes of the present invention.

“MOI” for purposes of the present invention means molecules of interest.The molecules of interest is preferably an enzyme, but is not limitedand can include bioparticles, proteins, or small molecules. Any materialuseful in being conjugated and immobilized to the AFOMC can be used forpurposes of the present invention and is included in the definition ofMOI.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the catabolic degradation of atrazine by P. ADP(Martinez et al., 2001).

FIG. 2. Conjugation of enzyme AtzA to nuetravidin.

FIG. 3. Biotinylation of amide-functionalized ordered mesoporous carbon(AFOMC).

FIG. 4. Conjugation of biotinylated functionalized ordered mesoporouscarbon (FOMC) to nuetravidinated purified enzymes AtzA.

FIG. 5. Radiochromatograms of ¹⁴C labeled atrazine reacted withamide-functionalized ordered mesoporous carbon conjugated with AtzAchlorohydrolase (A), and ¹⁴C labeled atrazine in control (reacted withDI water) for 2 hours (B).

FIG. 6. Radiochromatograms of ¹⁴C labeled atrazine reacted withamide-functionalized ordered mesoporous carbon conjugated with AtzAchlorohydrolase (A), and ¹⁴C labeled atrazine in control (reacted withDI water) for 14 days (B).

FIG. 7. Hydrolytic conversion of ortho-nitrophenyl-β-galactoside (ONPG)by 10 mg of amide-functionalized ordered mesoporous carbon (AFOMC)immobilized with β-galactosidase in 200 ml of 1M ONPG solution.

FIG. 8. Hydrolytic conversion of ortho-nitrophenyl-β-galactoside (ONPG)by amide-functionalized ordered mesoporous carbon (AFOMC) immobilizedwith β-galactosidase as a function of concentrations of AFOMC-basedbiocatalyst.

FIG. 9. Hydrolytic conversion of ortho-nitrophenyl-β-galactoside (ONPG)by the flow-through column packed with freshly prepared (A) and 6-weekold (B) amide-functionalized ordered mesoporous carbon (AFOMC)immobilized with β-galactosidase. The eluate was collected into eachfraction.

DETAILED DESCRIPTION OF INVENTION

The present invention provides for compositions and method of use foramide-functionalized ordered mesoporous carbon (AFOMC) as a vehicle todeliver material of interest (“MOT”) to a preferred material or target.Preferably, the MOT are enzymes that degrade pollutants or toxins toaide in remediation and cleaning of the material or target. The AFOMC ofthe present invention comprise at least one MOI conjugated to the AFOMC.Preferably, the AFOMC of the present invention comprise at least oneenzyme conjugated to the AFOMC capable of removing a pollutant or toxinfrom a source.

A method for creating the AFOMC of the present invention is alsoprovided. The method comprises the steps of synthesis of an orderedmesoporus carbon, purifying the MOI, and conjugating and immobilizingthe MOI to the surface of the AFOMC whereby allowing the AFOMC to thenbe delivered to a material or target. In a preferred embodiment, themethod of making the AFOMC of the present invention include the steps ofsynthesis of an ordered mesoporus carbon, producing and purifying thetarget enzyme, and conjugating and immobilizing the enzyme on theordered mesoporus carbon. Preferably, the method comprises the steps ofchemically modifying the amine residues on the AFOMC via use of SHTH tochemically conjugate biotin in its binding pockets; chemicallyconjugating to neutravidin via the C6 SFB chemistry; using purifiedenzyme to chemically conjugate to nuetravidin via the C6 SFB chemistry;and binding the biotinylated AFOMC to the nuetravidinated purifiedenzyme.

A method of creating a filter or biofilter and the resultant product isalso provided by the present invention. The biofilter preferablycomprises a porous material and the AFOMC of the present invention.Preferably, the MOI bound to the AFOMC are exposed to the material beingguided through the biofilter, such that pollutants and toxins areremoved from the material being guided through the biofilter as itpasses. Preferably, the material being guided through the biofilter is aliquid, more preferably, the liquid is water. However, any materialcapable of passing through a filter that may benefit from the removal ofpollutants or toxins will work for purposes of the present invention.

Any MOI that is beneficial in removing pollutants or toxins from amaterial will work for purposes of the present invention. Preferably,the MOI is an enzyme. Any enzymes that break down pollutants or toxinswill work for purposes of the present invention. One preferred class ofenzymes are atrazine-degrading enzymes. Atrazine-degrading enzymespreferably include, but are not limited to AtzA, AtzB, AtzC, AtzD, AtzE,AtzF, and combinations thereof. Another preferred class of enzymes arehydrolase enzymes. Hydrolase enzymes include, but are not limited toesterases, sugar hydrolases, ether bond hydrolases, proteases,carbon-nitrogen non-peptide hydrolases, acid anhydride hydrolases,carbon-carbon hydrolases, and combinations thereof. In a preferredembodiment, the hydrolase is a sugar hydrolases, and more preferably itis β-galactosidase. Additionally preferred MOIs include, but are notlimited to Xp1A cytochrome for removing RDX, PnrA for removing TNT, DfbBdioxin dioxenase for removing dioxin, ChRchromium reductase for reducingchromium 6+ to chromium 3+, and the like.

The AFOMC of the present invention can be used for air pollution, waterpollution, soil contamination, and radioactive pollution. Preferably,the present invention can be used to aid in air pollution, such as, butnot limited to, acid rain, air quality, chlorofluorocarbon, smog, andparticulates. With regard to water pollution, the present invention canhelp remove pollutants and toxins to help with, but not limited to, theenvironmental impact of pharmaceuticals and personal care products inthe water system, environmental monitoring of pollutants,eutrophication, hypoxia, marine debris, marine pollution, oceanacidification, oil spills, thermal pollutions, urban runoff, wastewater, water quality, and waterborne disease. Soil contamination thatcan be helped using the present invention includes, but is not limitedto bioremediation, phytoremediation, herbicides, pesticides, and soilguideline values. Some exemplary pollutants that can be removed usingthe present invention include, but are not limited to, atrazine,ortho-nitrophenyl-β-galactosidase (ONPG), chlorinated hydrocarbons,heavy metals, chromium, cadmium, lead, methyl tert-butyl ether (MTBE),zinc, arsenic, benzene, petrochemicals, carbon dioxide, greenhousegases, and the like.

In a preferred embodiment, the AFOMC of the present invention remove atleast 20% of the pollutants or toxins in the material exposed to theAFOMC, more preferably at least 30% are removed, still more preferablyat least 40% are removed, more preferably, at least 50% are removed,still more preferably at least 60% are removed, more preferably at least70% are removed, even more preferably at least 80% are removed, morepreferably at least 90% are removed, more preferably at least 95% areremoved, and most preferably 100% are removed. In a most preferredembodiment, in the case of a biofilter of the present inventioncontaining a hydrolase, about 60%-100% of the pollutants or toxins arehydrolyzed in a liquid being passed through a biofilter. In a mostpreferred embodiment, where the AFOMC of the present invention containan enzyme that breaks down atrazine, about 30% of the atrizine had beenremoved from a liquid in a time period of about 2 hours.

Additional Uses of the AFOMC of the present invention

As can be appreciated, the present invention has numerous uses acrossnumerous fields. Advantageously, the method of using the presentinvention is basically similar despite the field of use. In particular,the enzyme used and method of delivery may change depending on the use,but the basic platform does not. As the uses of the invention are toonumerous to enumerate herein, the scope of the invention should not belimited to the exemplary uses described herein.

Production of Biofuels

The AFOMC of the present invention may be used in the production ofbiofuels. It is contemplated that the MOI is any enzyme or combinationof enzymes capable of hydrolyzing starch, sucrose, lactose, cellulose orhemicelluloses into fermentable sugars. These sugars can be furtherfermented using enzymes capable of using the sugars to produce ethanol.The AFOMC can be delivered to a biomass such as agricultural crops, suchas corn, sugar cane and sugar beet, or from agricultural byproducts,such as whey and potato processing waste streams to aide in theproduction of ethanol.

Use of the AFOMC provides an improved production step for delivery ofthe desired enzymes. After the fuel and/or other are compounds producedthey can be recovered by suitable processing methods depending on theparticular material produced and the level of purity desired. Forexample, when producing ethanol the entire contents of the reaction canbe transferred to a distillation unit, and 96 percent ethanol/4 percentwater (by volume) can be distilled and collected. Fuel grade ethanol(99-100 percent ethanol) can be obtained by azeotropic distillation ofthe 96 percent ethanol, e.g., by the addition of benzene and thenre-distilling the mixture, or by passing the 96 percent ethanol throughmolecular sieves to remove the water.

Production of Biodiesel

The AFOMC system may be used in the production of biodiesel. Theconversion of vegetable oils to methyl- or other short chain esters in asingle transesterification reaction using lipases has led to theproduction of high-grade biodiesel. It is contemplated that the AFOMCwill incorporate an MOI that is a lipase. Exemplary lipases include,without limitation, lipases such as those from Pseudomonas cepacia,Rhizomocur miehei and Candida antarctica. One skilled in the art willrecognize that any lipase or combination of lipases could be used asMOIs with the AFOMC in the production of biodiesel. Use of the AFOMCprovides an improved production step for delivery of the lipases used inthe production of an energy source.

Bioremediation

The AFOMC of the present invention can be utilized in the remediation ofcontaminants. The AFOMC aides in the delivery of enzymes known in theart for the reduction of contaminants. Enzymes known in the art ofhaving the capability of breaking down or converting contaminants toless harmful substances can be used as MOIs with the AFOMC of thepresent invention. Suitable enzymes that may be used as MOIs, withoutlimitation, include mono- or di-oxygenases, reductases, dehalogenases,cytochrome P450 monoxygenases, enzymes involved in lignin metabolismsuch as laccases, lignin- and manganese peroxidases and bacterialphophotriesteraes. Suitable enzymes also include natural occurring,synthetic, and genetically engineered enzymes. By way of example, theenzyme AtzA produced by soil bacterium Pseudomonas strain ADP, iscapable of modifying the contaminant atrazine to the benign substancehydroatrazine (FIG. 20). By way of example the contaminated environmentscan include, but not limited to liquid environments, such as water,solid or semi-solid environments, such as soil, or gaseous environments,such as air. Exemplary contaminates include the following: polycyclicaromatic hydrocarbons (PAHs), polynitrated aromatic compounds,pesticides such as organochlorine insecticides, bleach-plant effluents,synthetic dyes, polymers, wood preservatives, chrysene, benzol[a]pyrene,coronene, dibenzothiophenes, cloro-dibenzofurans, cloro-dibenzop-dioxines, atrazine, lindane, polychlorinated biphenyl, syntheticpyrethroids, carbamates, and organophosphates to name a few. Exemplaryenzymes that may be used as MOIs include the following: mono- ordi-oxygenases, reductases, dehalogenases, cytochrome P450 monoxygenases,enzymes involved in lignin-metabolism such as laccases, lignin andmanganese peroxidases, and phosphotriesterases to name a few.

The AFOMC of the present invention may be combined with methods known inthe art for remediation. Suitable methods known in the art include,without limitation, bioremediation, vacuum or air stripping,immobilization, and soil washing-flushing. Immobilization is one of themore common methods, where solid matrices are introduced into the soilthat bind or otherwise minimize migration of the contaminate from theinitial site. Soil washing-flushing involves the introduction of aqueoussolution to the subsurface to mobilize the contaminates for treatment.It is contemplated that the AFOMC can be combined with soil-washingtechniques to introduce the BEMD particles to the subsurface. Also, BEMDparticles can be mixed into soil slurry and added to soil orincorporated into the desired environment through the use of soiltillage.

Fuel Cell

The AFOMC of the present invention can be utilized in fuel cells. TheAFOMC aides in the delivery of enzymes known in the art for theproduction of an energy source. Enzymes known in the art of having thecapability of breaking down organic material can be used as MOIs withthe AFOMC of the present invention. AFOMCs expressing one or more ofsuch enzymes as MOIs can be used with devices that directly convertbiocatalyst power generated from the degradation of organic matter intoelectrical energy. Exemplary enzymes include without limitationhydrogenases, laccases and other redox enzymes that have application aselectrocatalysts. In the field of biofuel cells, hydrogenases have beendemonstrated that convert hydrogen to generate an electric current andpossess similar energy conversion efficiency to noble-metal-basedcommercial methods. Laccases have also been incorporated into the designof biofuel cells since they are one of the few enzymes that can acceptelectrons from the cathodic compartment of a biofuel cell.

By way of example and without limitation, the AFOMC can be contactedwith environments containing organic material (i.e. biomass) such aswastewater and other undesirable substrates. As the enzymes delivered bythe AFOMC degrade the organic material through oxidation, hydrolysis,and other degradation methods, the fuel cell device converts this powerinto electricity. Fuel cell devices are known in the art such as thosedescribed in U.S. Patent Application No. 20100178530, incorporatedherein by reference. Several studies on electricity production fromartificial or real domestic wastewater, animal wastewater, foodwastewater, and recently hydrolysate from corn stover biomass has beenconducted and for this purpose several different types of fuel cellshave been developed both for batch and continuous mode operations.

Biohydrogen

The AFOMC of the present invention can be utilized in the production ofmolecular hydrogen as a renewable, efficient and pollution-free energysource. Hydrogen is colorless, odorless, tasteless, non-toxic and, oncombustion, it produces water as the only by-product. Hydrogen obtainedfrom biomass has the potential to compete with hydrogen produced byother methods such as from natural gas, which requires the catalyticconversion of hydrocarbons or electrochemical or photochemical watersplitting. Enzymes known in the art as hydrogenases can be used as MOIswith the AFOMC of the present invention. AFOMCs expressing one or moreof such enzymes as MOIs can be used for the production of hydrogen, forexample, by fermentation of sugar or, more preferably, from waste.

Biofilm Removal

Naturally occurring biofilms are continuously produced and oftenaccumulate on numerous industrial surfaces and on biological surfaces.In an industrial setting, the presence of these biofilms causes adecrease in the efficiency of industrial machinery, requires increasedmaintenance, and presents potential health hazards. For example, thesurfaces of water cooling towers become increasingly coated withmicrobially produced biofilm slime which both constricts water flow andreduces heat exchange capacity. Water cooling tower biofilms may alsoharbor pathogenic microorganisms such as Legionella pneumophila. Foodpreparation lines are routinely plagued by biofilm build-up both on themachinery and on the food product where biofilms often include potentialpathogens. Industrial biofilms are complex assemblages of insolublepolysaccharide-rich biopolymers which are produced and elaborated bysurface dwelling microorganisms. The chemical composition of industrialbiofilms are diverse and are specific to each species of surfacedwelling microorganism.

On a biological surface, the presence of these biofilms results in thegrowth of, and subsequent colonization by, pathogenic microorganisms onan internal or external surface of a host animal or on the surface ofobjects introduced into the animal (e.g. surgical implants). Animalpathogens which colonize surfaces are often maintained and protected byunique polysaccharide rich biofilms produced by the pathogen. Suchbiofilms coat the infected or colonized surface of the animal orimplanted object and continue to be produced during the disease process.For many diseases, biofilms are required for the disease process tobecome established and to progress. The chemical compositions ofpathogen-associated surface biofilms, which consist of complex mixturesof biopolymers, are specific to each species of pathogen.

The AFOMC can be utilized to treat and remove biofilms. Enzymes known inthe art as hydrolytic enzymes can be used as MOIs with the AFOMC of thepresent invention. AFOMCs expressing one or more of such enzymes as MOIscan be delivered to biofilm environments such that the hydrolyticenzymes significantly degrade or remove the biofilm. Techniques areknown in the art for biofilm removal such as those described in U.S.Patent Application No. 20100159563, incorporated herein by reference.

Drug Delivery

The AFOMC of the present invention may be utilized in deliveringtherapeutic molecules to a subject. Molecules such as therapeuticproteins may be used as MOIs with the AFOMC. These bioparticles may beadministered to a subject by methods known in the industry or describedherein. Suitable therapeutic proteins include those known in the art andthose yet to be discovered.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs at the time of filing.

EXAMPLES Example 1

This example illustrates synthesis and functionalization of orderedmesoporus carbon

Materials and Methods.

The mesoporous silica template (SBA-15) and ordered mesoporous carbon(OMC) were synthesized with the procedures described by Gu et al. (Gu,2007). The SBA-15 template for the synthesis of OMC consists of uniformarray of carbon rods arranged in hexagonal pattern (Hartmann, 2005).Synthesized OMC subsequently was treated with nitric acid, subsequentlychlorinated with thionyl chloride (SOCl₂), then functionalized withethylenediamine (EDA) (Gu, 2007; Tamai, 2006b; Zhu, 2011). Two and halfgrams of OMC were treated with 25 mL of 4M nitric acid (HNO₃) at 50′Cfor 2 hours. The HNO₃-treated activated carbon was chlorinated with 5%thionyl chloride (SOCl₂) in 20 mL of toluene solution at 70′C for 6 hrs.The immobilization of diamine on OMC was achieved by treating thechlorinated OMC with 0.05-Methylenediamine dissolved in 40 mL in toluenefor 4 hours. The functionalized OMC was collected by filtration andwashed by toluene for 2 hours to remove free EDA from carbon. Theresulting functionalized ordered mesoporous carbon was oven-driedovernight at 40′C under vacuum (Yantasee et al., 2004).

Enzyme Production and Purification

Total chromosomal DNA was isolated from an overnight culture ofPseudomonas sp. strain ADP grown in R media as described in Thompson etal. 2010. DNA was measured spectrophotometrically and utilized astemplate DNA for PCR amplification of the atzA gene.

A) The Purification of Recombinant Protein

PCR primers were designed for atzA open reading frame with promoterregion at the upstream. The amplicons with correct gene size were clonedinto the cloning vector pSC-A (Stratagene) and correct clones screenedvia restriction enzyme digestion, agarose electrophoresis and DNAsequencing analysis.

A 5-ml overnight culture of E. coli containing pSC-AtzA was used toinoculated 1 L of LB with ampicillin 100 μg/ml for protein expression.The culture preparation was incubated at 37′C with shaking and monitoreduntil OD600 reached 0.8. The culture for protein expression washarvested by centrifugation at 5000 rpm for 20 min and the cell pelletswere collected. The cell pellets were re-suspended with the solution of25 mM MOPS, pH6.9, and bead-beaten for 2 min at maximum speed at 4′C.The supernatant was collected as crude extract, after the bead-beatenpreparation was micro-centrifuged at maximum speed for 90 min at 4′C.The ammonium sulfate precipitation was performed with the crude extractfor 20% saturation. The precipitation preparation was centrifuged andthe pellets were collected and re-suspended with the solution of 25 mMMOPS, pH6.9. The re-suspension was dialyzed against the solution of 25mM MOPS, pH6.9 at 4′C to eliminate the remaining ammonium sulfate. Thepurity of the AtzA protein was determined by SDS-PAGE and Coomassie Bluestaining procedure as 40% at this point. The protein concentration ofthe protein preparation was determined by NanoDrop ND-1000spectrophotometer.

B) The Construction and Expression of Recombinant Protein with His Tagfor Purification

PCR primers were created that contained both a 5′ overhang and 3′overhang bearing part of the T7 promoter elements with a N-terminal6×His Tag and T7 terminator regions, respectively. The amplicon was thensubjected to PCR purification with a PCR purification kit (Qiagen), andused as template DNA for a second PCR reaction which annealed theremainder of the T7 promoter elements and T7 terminator. PCR primersutilized are described in Table 1. Correct amplicons were cloned intothe cloning vector pSC-A (Stratagene) and correct clones screened viarestriction enzyme digestion and DNA sequencing at the University ofMissouri DNA core.

TABLE 1 PCR Primers. Without 6xHis 5′ AtzAaggaggtagatacatgcaaacactcagcatccag 3′ AtzAggggttatgctatcactattattactagaggctgcgccaagctgg T7 Promoteratattatacgactcactatagggagataaggaggtagatac T7 Terminatoratatcaaaaaacccctcaagacccgtttagaggccccaaggggttatgct With 6xHis 5′ HisAtzA aggaggtagatacatgcatcaccatcaccatcaccaaacgctcagcatcc 3′ AtzAggggttatgctatcactattattactagaggctgcgccaagctgg T7 Promoteratattaatacgactcactatgggagataaggaggtagatac T7 Terminatoratatcaaaaaacccctcaagaccgtttagaggccccaaggggttatgct

AtzA protein expression was initiated using an in vitro T7 S30 TNT(transcription and translation) protein expression system (Promega).Briefly, 1 μg of atzA plasmid DNA was mixed with kit components as perthe manufacturer's recommendations. The reaction mixture was incubatedat 37° C. for 1 hour to allow for the transcription/translation reactionto occur. The reaction was stopped on ice for 1 minute. The totalprotein was then mixed 1:1 in 2×His column Binding buffer (ZymoResearch). After mixing, the protein mixture was purified via the H ispurification kit (Zymo Research) as per the manufacturers suggestion.The purified protein was subjected to dialysis overnight against 25 mMMOPS buffer, pH6.9 with one change of buffer. The resultant protein pool(pure AtzA) was analyzed via a NanoDrop ND-1000 spectrophotometer forprotein concentration. Protein purity was determined via separation of50 μg of AtzA protein on a 4-12% Tris-Glycine precast gel (Bio-rad)subjected to Coomassie blue staining analysis. The protein purity wasdetermined to be >95% AtzA for all reactions. The purified AtzA was usedimmediately or stored at −20° C. until needed.

Conjugation and Immobilization of Enzymes on the Functionalized OrderedMesoporous Carbon

Purified AtzA or β-galactosidase (Thermo-Pierce) was diluted in MOPS orPBS, respectively, to a concentration of 5 μg/μl. N-succunumidlyS-acetylthioacetate (SATA) solution was prepared by mixing 2 mg ofN-succunumidly S-acetylthioacetate (SATA) in 200 μl of dimethylformamide(DMF). Twenty μl of the SATA solution was added to 1 ml of targetprotein prep to conjugate the SATA groups onto the free amine groups ofthe AtzA/β-galactosidase enzymes. This reaction occurred for 30 minutesat room temperature. As shown in FIG. 1, the SATA-conjugated aminegroups of AtzA/β-galactosidase were then reduced (deacetylated) byreacting with hydroxylamine HCL in Maleimide Conjugation Buffer(Thermo-Pierce). After 2 hours of chemical reduction, the proteinpreparations (5 mg/ml) were separated from the free SATA groups andhydroxylamine via desalting columns (Thermo-Pierce). The nuetravidinatedpurified enzymes AtzA was accomplished by mixing 1 ml of Nuetravidin inMaleimide Conjugation Buffer (Thermo-Pierce, 5 mg/ml) in equal volume ofthe reduced SATA-modified enzymes incubated overnight at 4′C (FIG. 2).Centricon centrifugation filters (3,000 MWCO) were utilized to exchangethe enzymes into MOPS or PBS buffer for AtzA or (3-galactosidase,respectively.

Amide-functionalized ordered mesoporous carbon (AFOMC, 5 mg/ml) wasconjugated via its exposing amine groups to biotin using the EZ-LinkSulfo-NHS-LC kit (Thermo-Pierce) (FIG. 3). The nuetravidinated purifiedenzymes were then added to the biotinylated AFOMC in the ratio of 1:5 toallow the nuetravidin-biotin binding occurred (FIG. 4).

Chemical Analysis

One hundred microliters of atrazine solution with mixture of ¹⁴C-labeledatrazine and non-labeled atrazine was applied into 1 mL of bufferscontaining 10 mM KCl in 25 mM (MOPS) buffer (pH 6.9) to achieved thetotal atrazine concentration of 2000 ppb (μg/L) with radioactivity 0.1μci/mL. One hundred microlilters of AFOMC conjugated with AtzA enzymes(5 mg/ml) was added into the buffer solution containing atrazine andincubated at room temperature 25° C. The samples were injected into aShimadzu SCL-10Avp high performance liquid chromatography system (HPLC)(Columbia, Md.). ¹⁴C-ATR and its degradation products were separatedusing a silica-based Columbus C8 column (4.6 mm×250 mm, 5 μm;Phenomenex, Torrance, Calif.). The radioactivity was detected by anin-line IN/US ScinFlow β-Ram Model 3 (Tampa, Fla.) flow scintillationanalyzer (HPLC-FSA). Injection volume was 10 μL, and mobile phase flowrate was 1 mL min-1. The ¹⁴C-ATR and its metabolites were eluted with atwo-part mobile phase gradient. Mobile phase A consisted of 0.1% H₃PO₄buffer (pH=2.1), and mobile phase B was 100% ACN. The gradient startedat 10% and ramped linearly to 40% at 30 min, 75% at 40 min, 10% at 45min, and held at 10% for 14 min. Metabolites were identified bycomparing the retention times of unlabeled standards based on HPLC-UVdetection at 220 nm. The standards including atrazine (ATR),deethylatrazine (DEA), deisopropylatrazine (DIA), hydroxyatrazine (HA),deisopropylhydroxyatrazine (DIHA), deethylhydroxyatrazine (DEHA),didealkylatrazine (DDA), ammeline (AM) and Ammelide were purchasedthrough ChemService (West Chester, Pa.).

To determine the β-galactosidase activity, theortho-Nitrophenyl-β-galactoside (ONPG) was used as subtract. Theconcentrations of free ortho-nitrophenol as a result of the hydrolyzedortho-Nitrophenyl-β-galactoside were measured spectrophotometrically at420 nm wavelength.

Results and Discussion

N-Hydroxysulfosuccinimide (Sulfo-NHS) ester of biotin was used forconjugate the NHS-activated biotin with the FOMC. The primary amine asresults of the amide functionalization reaction reacts efficiently withthe NHS-activated biotin (FIG. 2). As shown in FIG. 5, thechlorohydrolaseAtzA immobilized on the surface of functionalized orderedmesoporous carbon still retained it enzymatic activity. It transformedabout 30% of atrazine in the solution into less toxic and less mobilehydroxyatrazine within 2 hours. On the other hand, the concentration ofatrazine in the control treatment was constant in the solutionthroughout the experimental period. It should be noted that, thehalf-life of atrazine is about 350 days under the sterilized conditionin this studies. Therefore, we did not expect to observe any degradationof atrazine in the control treatment.

The decreased in total radioactivity in the solution over time as shownin FIG. 6 suggested the high specific surface area of the AFOMC providea large adsorption capacities for substrate atrazine and its metabolitehydroxyatrazine through electrostatic interactions and/or covalentbindings. The AFOMC naturally draws in charged molecules, such as theherbicide atrazine, towards the attached enzymes that will convert theatrazine to benign hydroxyatrazine. This system can be used with avariety of enzymes and pollutants. We have utilized this invention todesign a biofilter that removes atrazine from stagnant and free flowingwater sources and have proven the concept in small scale laboratoryexperiments. In addition, immobilization of multiple ATR-degradingenzymes, such as combination of AtzA, AtzB, AtzC, AtzD, AtzE, and AtzF,may accelerate the degradation and transformation processes.

This system can be used with a variety of enzymes to mitigate thecontamination of both organic and inorganic pollutants. As shown in theFIG. 7, more than 95% of the ortho-nitrophenyl-β-galactoside (ONPG) washydrolyzed by the AFOMC immobilized with β-galactosidase during 12 hoursof reaction period. As we expected, the hydrolysis of ONPG byamide-functionalized ordered mesoporous carbon (AFOMC) immobilized withβ-galactosidase was significantly (p<0.005) enhanced with the increasingconcentrations of AFOMC-based biocatalyst (FIG. 8).

We have utilized this invention to design a biofilter that removesatrazine and other class of pollutants from stagnant and free flowingwater sources and have proven the concept in small scale laboratoryexperiments. As the results shown in FIG. 9A, when the substratenitrophenyl-β-galactoside ONPG passed through the flow-through columnpacked with AFOMC immobilized with β-galactosidase, 80% to 100% of theONPG in the effluent was rapidly hydrolyzed. The results also suggestedthat the column packed with freshly prepared AFOMC-based biocatalystsoutperformed the system packed with 6-weeks old AFOMC-based biocatalysts(FIG. 9B).

Other Possible Uses

This conjugation of bioactive enzymes onto the amide-functionalizedordered mesoporous carbon have a wide range of other commercialapplications for development biocatalysts, biofilters (e.g., Xp1Acytochrome P450 for removing RDX, PnrA for removing TNT, DbfB dioxindioxenase for removing dioxin, ChrR chromium reductase for reducingchromium 6+ to chromium 3+), fuel cell (enzyme based biological fuelcell), imaging (antibodies and fluorescent proteins), biofuelproduction, drug delivery systems (e.g., antimicrobial proteins:lysozyme etc), medical therapeutics and biosensors.

While the invention has been described in connection with specificembodiments thereof, it will be understood that the inventive device iscapable of further modifications. This patent application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure as come within known or customarypractice within the art to which the invention pertains and as may beapplied to the essential features herein.

REFERENCES

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The invention claimed is:
 1. A contaminate reduction compositioncomprising: a) an amide-functionalized ordered mesoporous carbon(AFMOC); conjugated with b) a biotin conjugate; and, c) atoxin-degrading enzyme.
 2. The contaminate reduction composition ofclaim 1, wherein the toxin-degrading enzyme degrades atrazine.
 3. Thecontaminate reduction composition of claim 1, wherein the biotinconjugate is chemically associated with the amide-functionalized orderedmesoporous carbon.
 4. The contaminate reduction composition of claim 1,wherein the toxin-degrading enzyme is capable of binding to the biotinconjugate.